Adoptive Cell Therapy Of Cancer Research Articles

STING-driven activation of T cells: relevance for the adoptive cell therapy of cancer

Adoptive cell therapy (ACT) can successfully treat hema-topoietic cancers but lacks efficacy against solid tumors. This is due to insufficient T cell infiltration, high tumor heterogeneity, frequent antigen loss with subsequent tumor escape, and the immunosup-pressive tumor microenvironment (TME). Alternative methods to boost the anticancer efficacy of adoptively transferred cells are ac-tively pursued. Among adjuvants that are utilized to stimulate anti-cancer immune responses, ligands of the stimulator of interferon genes (STING) pathway have received increasing attention. STING activation can trigger dendritic cell (DC) activation and endogenous immune responses, thereby preventing tumor escape. Activation of the STING pathway in the context of ACT was accordingly associated with improved T cell trafficking and persistence in the TME com-bined with the reduced presence of immunosuppressive cells. Re-cent findings also suggest cell-intrinsic effects of STING ligands on T cells. Activation of the STING signaling pathway was in this regard shown to enhance effector functions of CD4+ and CD8+ T cells, sug-gesting that the STING signaling could be exploited to harness T cell anticancer functions. In this review, we will discuss how the STING signaling can be used to enhance the anticancer efficacy of ACT.

The effect of physical exercise on anticancer immunity.

Regular physical activity is associated with lower cancer incidence and mortality, as well as with a lower rate of tumour recurrence. The epidemiological evidence is supported by preclinical studies in animal models showing that regular exercise delays the progression of cancer, including highly aggressive malignancies. Although the mechanisms underlying the antitumorigenic effects of exercise remain to be defined, an improvement in cancer immunosurveillance is likely important, with different immune cell subtypes stimulated by exercise to infiltrate tumours. There is also evidence that immune cells from blood collected after an exercise bout could be used as adoptive cell therapy for cancer. In this Perspective, we address the importance of muscular activity for maintaining a healthy immune system and discuss the effects of a single bout of exercise (that is, 'acute' exercise) and those of 'regular' exercise (that is, repeated bouts) on anticancer immunity, including tumour infiltrates. We also address the postulated mechanisms and the clinical implications of this emerging area of research.

Role and Potential of Different T Helper Cell Subsets in Adoptive Cell Therapy

Historically, CD8+ T cells have been considered the most relevant effector cells involved in the immune response against tumors and have therefore been the focus of most cancer immunotherapy approaches. However, CD4+ T cells and their secreted factors also play a crucial role in the tumor microenvironment and can orchestrate both pro- and antitumoral immune responses. Depending on the cytokine milieu to which they are exposed, CD4+ T cells can differentiate into several phenotypically different subsets with very divergent effects on tumor progression. In this review, we provide an overview of the current knowledge about the role of the different T helper subsets in the immune system, with special emphasis on their implication in antitumoral immune responses. Furthermore, we also summarize therapeutic applications of each subset and its associated cytokines in the adoptive cell therapy of cancer.

Preclinical In Vitro and In Vivo Models for Adoptive Cell Therapy of Cancer

Adoptive cellular therapies are making major strides in the treatment of cancer, both for hematologic and solid tumors. These cellular products include chimeric antigen receptor T cells and T-cell receptor-modified T cells, tumor-infiltrating lymphocytes, marrow-infiltrating T cells, natural killer cells as well as macrophage-based therapeutics. Advancement in genomics, computational biology, immunology, and cell therapy manufacturing has facilitated advancement of adoptive T cell therapies into the clinic, whereas clinical efficacy has driven Food and Drug Administration approvals. The growth of adoptive cellular therapy has, in turn, led to innovation in the preclinical models available, from ex vivo cell-based models to in vivo xenograft models of treatment. This review focuses on the development and application of in vitro models and in vivo models (cell line xenograft, humanized mice, and patient-derived xenograft models) that directly evaluate these human cellular products.

Allogeneic Tumor Antigen-Specific T Cells for Broadly Applicable Adoptive Cell Therapy of Cancer.

T cells specific for major histocompatibility complex (MHC)-presented tumor antigens are capable of inducing durable remissions when adoptively transferred to patients with refractory cancers presenting such antigens. When such T cells are derived from healthy donors, they can be banked for off-the-shelf administration in appropriately tissue matched patients. Therefore, tumor antigen-specific, donor-derived T cells are expected to be a mainstay in the cancer immunotherapy armamentarium. In this chapter, we analyze clinical evidence that tumor antigen-specific donor-derived T cells can induce tumor regressions when administered to appropriately matched patients whose tumors are refractory to standard therapy. We also delineate the landscape of MHC-presented and unconventional tumor antigens recognized by T cells in healthy individuals that have been targeted for adoptive T cell therapy, as well as emerging antigens for which mounting evidence suggests their utility as targets for adoptive T cell therapy. We discuss the growing technological advancements that have facilitated sequence identification of such antigens and their cognate T cells, and applicability of such technologies in the pre-clinical and clinical settings.

Detection and viability of murine NK cells in vivo in a lymphoma model using fluorine-19 MRI.

Natural killer (NK) cell therapies are being increasingly used as an adoptive cell therapy for cancer because they can recognize tumor cells in an antigen-independent manner. While promising, the understanding of NK cell persistence, particularly within a harsh tumor microenvironment, is limited. Fluorine-19 (19 F) MRI is a noninvasive imaging modality that has shown promise in longitudinally tracking cell populations in vivo; however, it has not been studied on murine NK cells. In this study, the impact of 19 F labeling on murine NK cell viability and function was assessed in vitro and then used to quantify NK cell persistence in vivo. While there was no noticeable impact on viability, labeling NK cells with 19 F did attenuate cytotoxicity against lymphoma cells in vitro. Fluorescent microscopy verified 19 F labeling in both the cytoplasm and nucleus of NK cells. Lymphoma-bearing mice were given intratumoral injections of 19 F-labeled NK cells in which signal was detectable across the 6 day observation period via 19 F MRI. Quantification from the composite images detected 78-94% of the initially injected NK cells across 6 days, with a significant decrease between Days 3 and 6. Postmortem flow cytometry demonstrated retention of 19 F intracellularly within adoptively transferred NK cells with less than 1% of 19 F-containing cells identified as tumor-associated macrophages that presumably ingested nonviable NK cells. This work demonstrates that 19 F MRI offers a specific imaging platform to track and quantify murine NK cells within tumors noninvasively.

Recruitment, Infiltration, and Cytotoxicity of HLA-Independent Killer Lymphocytes in Three-Dimensional Melanoma Models.

Simple SummaryLimited therapeutic results of immune checkpoint inhibitors in definite tumor settings, such as melanoma, call for alternative or complementary approaches. Among these, adoptive cell therapy (ACT) by means of HLA-independent tumor killer lymphocytes is a promising approach. We aimed at developing a pre-clinical 3D model to investigate and visualize the interaction between tumor and immune effectors in melanoma. To this aim, we employed Cytokine-Induced Killer cells (CIK) and NK-92 on patient-derived melanoma samples. By means of imaging-based methods, we measured the effector recruitment on the 3D targets, their infiltration, and cytotoxic activity. Our results and methodologies can be easily generalized to other effectors and other classes of tumors and help elucidate fundamental questions on the basic biology and kinetics of immune effector recruitment in a realistic 3D setting mimicking a solid tumor.Cancer adoptive cell therapy (ACT) with HLA-independent tumor killer lymphocytes is a promising approach, with intrinsic features potentially addressing crucial tumor-escape mechanisms of checkpoint inhibitors. Cytokine-induced Killer (CIK) and Natural Killer (NK) lymphocytes share similar tumor-killing mechanisms, with preclinical evidence of intense activity against multiple solid tumors and currently testing in clinical studies. To improve the effective clinical translation of such ACT approaches, several fundamental questions still need to be addressed within appropriate preclinical contexts, capable of overcoming limitations imposed by most traditional two-dimensional assays. Here, we developed a novel experimental approach to explore, dissect, and visualize the interactions of CIK and NK lymphocytes with melanoma tumors in vitro in 3D. Primary melanoma cells were assembled into small tumors that were dispersed in a 3D matrix and challenged with patient-derived CIK or the NK-92 cell line. By means of imaging-based methods, we reported, visualized, and quantitatively measured the recruitment of CIK and NK on the 3D targets, their infiltration, and cytotoxic activity. Our results support the effective tumor recruitment and tumor infiltration by CIK and NK. Such features appeared dependent on the specific geometric aspects of the environment but can be explained in terms of directional migration toward the tumor, without invoking major feedback components. Overall, our 3D platform allows us to monitor the processes of tumor recruitment, infiltration, and killing by means of live measurements, revealing important kinetic aspects of ACT with CIK and NK against melanoma.

Applications of CRISPR Genome Editing to Advance the Next Generation of Adoptive Cell Therapies for Cancer.

Adoptive cell therapy (ACT) for cancer shows tremendous potential; however, several challenges preclude its widespread use. These include poor T-cell function in hostile tumor microenvironments, a lack of tumor-specific target antigens, and the high cost and poor scalability of cell therapy manufacturing. Creative genome-editing strategies are beginning to emerge to address each of these limitations, which has initiated the next generation of cell therapy products now entering clinical trials. CRISPR is at the forefront of this revolution, offering a simple and versatile platform for genetic engineering. This review provides a comprehensive overview of CRISPR applications that have advanced ACT. SIGNIFICANCE: The clinical impact of ACT for cancer can be expanded by implementing specific genetic modifications that enhance the potency, safety, and scalability of cellular products. Here we provide a detailed description of such genetic modifications, highlighting avenues to enhance the therapeutic efficacy and accessibility of ACT for cancer. Furthermore, we review high-throughput CRISPR genetic screens that have unveiled novel targets for cell therapy enhancement.

Highly Efficient Generation of Transgenically Augmented CAR NK Cells Overexpressing CXCR4.

Natural killer (NK) cells are a noteworthy lymphocyte subset in cancer adoptive cell therapy. NK cells initiate innate immune responses against infections and malignancies with natural cytotoxicity, which is independent of foreign antigen recognition. Based on these substantive features, genetically modifying NK cells is among the prime goals in immunotherapy but is currently difficult to achieve. Recently, we reported a fully human CAR19 construct (huCAR19) with remarkable function in gene-modified T-cells. Here, we show efficient and stable gene delivery of huCAR19 to primary human NK cells using lentiviral vectors with transduction efficiencies comparable to those achieved with NK cell lines. These huCAR19 NK cells display specific and potent cytotoxic activity against target cells. To improve homing of NK cells to the bone marrow, we augmented huCAR19 NK cells with the human CXCR4 gene, resulting in transgenically augmented CAR NK cells (TRACKs). Compared to conventional CAR NK cells, TRACKs exhibit enhanced migration capacity in response to recombinant SDF-1 or bone marrow stromal cells while retaining functional and cytolytic activity against target cells. Based on these promising findings, TRACKs may become a novel candidate for immunotherapeutic strategies in clinical applications.

Artificial feeder cells expressing ligands for killer cell immunoglobulin-like receptors and CD94/NKG2A for expansion of functional primary natural killer cells with tolerance to self

Background aimsNatural killer (NK) cells are promising cells for immunotherapy of cancer, and there are ongoing efforts to improve their ex vivo expansion to clinically relevant numbers. This study focused on the development of a C1-, C2-, Bw4 killer cell immunoglobulin-like receptor (KIR) ligand and NKG2A ligand-containing feeder cell line for autonomous expansion of functional NK cells. MethodsPC3PSCA-derived feeder cells expressing IL-2, 4-1BBL and membrane-bound IL-15-mutDAP12 (mIL-15d) fusion protein in combinations or alone were generated and used for expansion. Expanded NK cells were analyzed with respect to subpopulations, expression of NK cell receptors and immune checkpoint molecules as well as their cytotoxicity against K562 cells, cetuximab-marked tumor cells and autologous B cells. ResultsOnly combinatorial expression of IL-2 plus 4-1BBL or IL-2, 4-1BBL plus mIL-15d in feeder cells efficiently expanded NK cells and supported selective outgrowth of NK cells from peripheral blood mononuclear cell samples. Best expansion of NK cells was achieved using PC3PSCA-IL-2-4-1BBL-mIL-15d feeder cells. Such expanded NK cells exhibited upregulation of natural cytotoxicity receptors, DNAM-1 and NKG2C and induced expression of high affinity IL-2 receptor, which were paralleled by attenuated KIR and increased expression of NKG2A and ILT2. In addition, elevated TIM-3 levels were noted and PD-1 and T cell immunoreceptor with Ig and ITIM domain (TIGIT) levels remained low. Expanded NK cells were highly cytolytic when encountering K562 cells and cetuximab-marked target cells but remained unresponsive to autologous B cells and target cells with protective levels of human leukocyte antigen. ConclusionsCollectively, the results demonstrate the feasibility of PC3PSCA-IL-2-4-1BBL-mIL-15d feeder cells for robust expansion of NK cells, which remain tolerant to self and could be used in the future for adoptive cell therapy of cancer.

Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy.

Adoptive cell therapy represents a new paradigm in cancer immunotherapy but can be limited by poor persistence and function of transferred T cells1. Here, through an in vivo pooled CRISPR-Cas9 mutagenesis screening, we demonstrate that CD8+ T cells are reprogrammed to long-lived effector cells with extensive accumulation, better persistence and robust effector function in tumors by targeting Regnase-1. Regnase-1-deficient CD8+ T cells show markedly improved therapeutic efficacy against mouse melanoma and leukemia. Through a secondary genome-scale CRISPR-Cas9 screening, we identify BATF as the key target of Regnase-1 and a rheostat in shaping antitumor responses. Loss of BATF suppresses the elevated accumulation and mitochondrial fitness of Regnase-1-deficient CD8+ T cells. Conversely, we reveal that targeting additional signaling factors including PTPN2 and SOCS1 improves the therapeutic efficacy of Regnase-1-deficient CD8+ T cells. Our findings suggest that T-cell persistence and effector function can be coordinated in tumor immunity and point to new avenues to improve the efficacy of adoptive cell therapy for cancer.

Clinical-Scale Production of CAR-T Cells for the Treatment of Melanoma Patients by mRNA Transfection of a CSPG4-Specific CAR under Full GMP Compliance.

Chimeric antigen receptor (CAR)-T cells already showed impressive clinical regressions in leukemia and lymphoma. However, the development of CAR-T cells against solid tumors lags behind. Here we present the clinical-scale production of CAR-T cells for the treatment of melanoma under full GMP compliance. In this approach a CAR, specific for chondroitin sulfate proteoglycan 4 (CSPG4) is intentionally transiently expressed by mRNA electroporation for safety reasons. The clinical-scale protocol was optimized for: (i) expansion of T cells, (ii) electroporation efficiency, (iii) viability, (iv) cryopreservation, and (v) potency. Four consistency runs resulted in CAR-T cells in clinically sufficient numbers, i.e., 2.4 × 109 CAR-expressing T cells, starting from 1.77x108 PBMCs, with an average expansion of 13.6x, an electroporation efficiency of 88.0% CAR-positive cells, a survival of 74.1% after electroporation, and a viability of 84% after cryopreservation. Purity was 98.7% CD3+ cells, with 78.1% CD3+/CD8+ T cells and with minor contaminations of 1.2% NK cells and 0.6% B cells. The resulting CAR-T cells were tested for cytolytic activity after cryopreservation and showed antigen-specific and very efficient lysis of tumor cells. Although our work is descriptive rather than investigative in nature, we expect that providing this clinically applicable protocol to generate sufficient numbers of mRNA-transfected CAR-T cells will help in moving the field of adoptive cell therapy of cancer forward.

The emerging role of γδ T cells in cancer immunotherapy.

The recent successes of chimeric antigen receptor T cells in the treatment of hematological malignancies have clearly led to an explosion in the field of adoptive cell therapy for cancer. Current efforts are focused on the translation of this exciting technology to the treatment of solid tumors and the development of allogeneic ‘off-the-shelf’ therapies. γδ T cells are currently gaining considerable attention in this field as their unique biology and established role in cancer immunosurveillance place them in a unique position to potentially overcome these challenges in adoptive cell therapy. Here, we review the relevant aspects of the function of γδ T cells in cancer immunity, and summarize clinical observations and clinical trial results that highlight their emerging role as a platform for the development of safe and effective cancer immunotherapies.

Abstract IA37: Enhancing the function of CAR T cells via a universal vaccine strategy

Abstract We recently developed a strategy to target peptide vaccines to lymph nodes, by linking peptide antigens to albumin-binding phospholipid-polymers. Small peptides are normally rapidly dispersed in the bloodstream following parenteral injection, but binding of amphiphile-peptides to endogenous albumin, which constitutively traffics from blood to lymph, retargeted these molecules to lymph nodes. However, these lipid-polymer conjugates bind to albumin with a relatively low affinity, and these molecules can also partition into cell membranes. We hypothesized that by attaching a small molecule, peptide, or protein ligand for a chimeric antigen receptor (CAR) to the same polymer-lipid tail (forming an “amph-vax” molecule), CAR ligands could be delivered efficiently to lymph nodes by albumin and subsequently partition into membranes of resident antigen presenting cells (APCs), thereby co-displaying a CAR T cell ligand from the cell surface together with native cytokine/receptor costimulation. In syngeneic mouse models of adoptive cell therapy, we demonstrated that this approach effectively concentrates CAR T ligands on the surfaces of dendritic cells in lymph nodes, leading to profound expansion of amph-vax-boosted CAR T cells in vivo. Amph-vax boosting safely increased the polyfunctionality of CAR T cells in parallel with T cell expansion. In a syngeneic model of melanoma, this converted a CAR T treatment that had no impact on tumor progression to a therapy that strongly delayed tumor outgrowth and enhanced survival. We have reduced to practice three different strategies to generalize this approach to any CAR of interest. This concept provides a strategy to regulate the expansion and function of CAR T cells directly in vivo to enhance adoptive cell therapy of cancer. Citation Format: Darrell J. Irvine. Enhancing the function of CAR T cells via a universal vaccine strategy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr IA37.

Abstract IA06: Enhancing combination immunotherapy and adoptive cell therapy of cancer via lymph node-targeted vaccines

Abstract Lymph node targeting is achieved clinically is sentinel lymph node mapping in cancer patients, where small-molecule dyes are efficiently delivered to lymph nodes by binding to serum albumin. To mimic this process in vaccine delivery, we synthesized amphiphile-vaccines (lipid-polymer conjugates of peptides, proteins, or molecular adjuvants) designed to non-covalently bind vaccine antigens and adjuvants to endogenous albumin. These amphiphile conjugates both protect peptide antigens from premature degradation and greatly increase their delivery to lymph nodes. Such “albumin-hitchhiking” amphiphiles elicited as much as 30-fold amplified cellular immune responses and antitumor immunity against multiple antigens, and further enable mucosal delivery of therapeutic vaccines through albumin-mediated transport across epithelial barriers via the neonatal Fc receptor. When combined with complementary immunotherapy agents, these lymph node-targeted vaccines are capable of eradicating large established tumors in several mouse models, providing a blueprint for curative immunotherapies. We have further demonstrated that amph-vaccines can be used to deliver ligands for chimeric antigen receptor (CAR) T cells to lymph nodes, creating efficient booster vaccines for CAR T cells. Citation Format: Darrell J. Irvine. Enhancing combination immunotherapy and adoptive cell therapy of cancer via lymph node-targeted vaccines [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr IA06.

Abstract 3586: A universal killer T-cell for adoptive cell therapy of cancer

Abstract T cell-mediated immunotherapy of cancer has achieved remarkable results in hard-to-beat cancers. The main challenge of adoptive T-cell transfer (ACT) is its labor intensive and costly production and logistics as well as its dependency on the quality of the patient's T cells. To overcome these hurdles we have designed a universal cell line for TCR expression by modifying the FDA-approved NK cell line, NK-92. Advantages of using this cell line is that it is easy to expand and can readily be genetically engineered. However, tumor cell recognition and killing by NK-92 is not antigen specific. This can be controlled by introducing an antigen receptor, such as a chimeric antigen receptor (CAR) or, as in the current work, a TCR. We herein present evidence that NK-92 can be modified to become a T cell-like lymphocyte which we named UK-92 cell (Universal Killer). UK-92 expressing a therapeutic TCR showed conserved binding capacity to the cognate pMHC. Phosphoflow cytometry results indicated that the introduced TCR was able to mediate intracellular signaling upon either crosslinking or cognate pMHC binding. Our data showed that both early and late TCR signalling players were activated in a TCR-specific manner (anti-CD3/anti-CD28 stimulation) and further in a pMHC specific manner. In vitro functional assays using TCRs isolated from both CD8 and CD4 T cells demonstrated that UK-92-TCR could be stimulated in a pMHC-specific manner and, importantly, could kill tumor cells specifically even in a tumor-like microenvironment such as spheroids. We have now shown in vitro that UK-92 cells are as specific and potent as redirected T cells to kill target cells. Finally, encouraging in vivo data showed that mice receiving UK92 cells expressing a therapeutic TCR experienced reduction in tumor load and enhanced survival compared with control mice. If confirmed, the use of UK92 as a universal cell line might pave the way to truly off-the-shelf therapeutic effector cells for cancer immunotherapy and leading to drastic reduction of cell production time, logistic and cost. Citation Format: Pierre Dillard, Nadia Mensali, June Helen Myklebust, Mickael Hebeisen, Gjertrud Skorstad, Marit Renee Myrhe, Anne Fåne, Gustav Gaudernack, Gunnar Kvalheim, Else Marit Inderberg, Sebastien Wälchli. A universal killer T-cell for adoptive cell therapy of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3586.

Advances in evidence-based cancer adoptive cell therapy.

Adoptive cell therapy (ACT) has been developed in cancer treatment by transferring/infusing immune cells into cancer patients, which are able to recognize, target, and destroy tumor cells. Recently, sipuleucel-T and genetically-modified T cells expressing chimeric antigen receptors (CAR) show a great potential to control metastatic castration-resistant prostate cancer and hematologic malignancies in clinic. This review summarized some of the major evidence-based ACT and the challenges to improve cell quality and reduce the side effects in the field. This review also provided future research directions to make sure ACT widely available in clinic.

Enhancing Adoptive Cell Therapy of Cancer through Targeted Delivery of Small-Molecule Immunomodulators to Internalizing or Noninternalizing Receptors.

Adoptive cell therapy (ACT) has achieved striking efficacy in B-cell leukemias, but less success treating other cancers, in part due to the rapid loss of ACT T-cell effector function in vivo due to immunosuppression in solid tumors. Transforming growth factor-β (TGF-β) signaling is an important mechanism of immune suppression in the tumor microenvironment, but systemic inhibition of TGF-β is toxic. Here we evaluated the potential of targeting a small molecule inhibitor of TGF-β to ACT T-cells using PEGylated immunoliposomes. Liposomes were prepared that released TGF-β inhibitor over ∼3 days in vitro. We compared the impact of targeting these drug-loaded vesicles to T-cells via an internalizing receptor (CD90) or noninternalizing receptor (CD45). When lymphocytes were preloaded with immunoliposomes in vitro prior to adoptive therapy, vesicles targeted to both CD45 and CD90 promoted enhanced T-cell expression of granzymes relative to free systemic drug administration, but only targeting to CD45 enhanced accumulation of granzyme-expressing T-cells in tumors, which correlated with the greatest enhancement of T-cell antitumor activity. By contrast, when administered i.v. to target T-cells in vivo, only targeting of a CD90 isoform expressed exclusively by the donor T-cells led to greater tumor regression over equivalent doses of free systemic drug. These results suggest that in vivo, targeting of receptors uniquely expressed by donor T-cells is of paramount importance for maximal efficacy. This immunoliposome strategy should be broadly applicable to target exogenous or endogenous T-cells and defines parameters to optimize delivery of supporting (or suppressive) drugs to these important immune effectors.

Abstract B53: T lymphocyte engineering with responsive cytokine nanogels for enhanced efficacy and safety of adoptive cell therapy for cancer

Abstract Adoptive cell transfer (ACT) with autologous tumor-reactive T-cells has recently shown great promise in the treatment of leukemias in clinical trials. However, effective control of solid tumors, in particular resistant, metastatic, or recurrent cancers, remain challenging. One of the major limitations is the rapid decline of T-cell function in the immunosuppressive tumor microenvironment. In order to maintain high numbers of viable antigen-specific cytotoxic T-cells in tumors, co-administration of supporting immunostimulant agents such as cytokines together with transferred cells is often employed. However, the high systemic doses of such agents needed to enhance T-cell functionality inside solid tumors can also result in serious side effects. Here, we developed a cell-responsive nanogel (NG) based on a novel carrier-free delivery strategy to effectively and specifically support adoptively transferred T-cells with recombinant cytokines such as interleukin-2 (IL-2) or interleukin-15 (IL-15). IL-2-Fc or an IL-15 superagonist (IL-15Sa) were chemically crosslinked with a disulfide linker to form these protein NGs, which were subsequently bound to and stably anchored on the plasma membrane of adoptively transferred T-cells ex vivo. These NGs had exceptionally high loading of cytokines (~70 wt%) and released native cytokine proteins in physiological condition in a sustained manner through breakdown of a degradable disulfide linker in response to the reduction activity at the plasma membrane surface of activated T-cells, and thus enabled continuous pseudo-autocrine stimulation of transferred CD8+ T-cells. In a mouse model of subcutaneous B16F10 melanoma tumors, adoptively transferred mouse pmel-1 CD8+ T-cells with optimized IL-15Sa-NGs showed markedly enhanced expansion and persistence in tumors and thus improved the anti-cancer efficacy. Quantification of transferred tumor-specific T-cells in tumors 7 days after transfer showed that surface-bound IL-15Sa-NGs resulted in ~16-fold more tumor-reactive T-cells compared to T-cells supported by an equivalent dose of soluble IL-15Sa administered systemically. Surface-bound IL-15Sa-NGs could be used to “backpack” T-cells with doses of cytokine that slowly released and maintained T-cell function over at least 2 weeks in vivo, while the same doses administered systemically as a bolus or repeated lower-dose injections were very toxic, eliciting systemic cytokine syndrome. These data demonstrate that T-cell surface-bound responsive cytokine-NGs provide autocrine support to the tumor-reactive T-cells to enhance efficacy of ACT therapy against solid tumors and safely administered potent adjuvant immunostimulants to donor T-cells. Citation Format: Li Tang, Yiran Zheng, Llian Mabardi, Darrell Irvine. T lymphocyte engineering with responsive cytokine nanogels for enhanced efficacy and safety of adoptive cell therapy for cancer. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B53.

A Novel NKp44-Based Chimeric Antigen Receptor That Targets Multiple Types of Cancer

Genetic modification of T cells with an artificial tumor-targeting chimeric antigen receptor (CAR) is a new approach for adoptive cell therapy for cancer. Defining cell surface molecules that are both selectively expressed on cancer cells and can be safely targeted with T cells or NK cells is a significant challenge in this research field.NKp44 is a member of the natural cytotoxicity receptor (NCR) families and also known as NCR2. Expression of NKp44 is limited to activating NK cells, which leads to a marked increase in cytotoxicity against tumors. The receptor contains one extracellular immunoglobulin domain, type I transmembrane (TM) domain, and intracellular (IC) domain, and its surface expression seems to require binding of the TM domain to adaptor molecules of DAP12 accessory protein that contains ITAMs. The ligand for NKp44 is considered damage-associated molecular pattern molecules, which have been reported to be expressed by various types of cancer cells but not by healthy cells. Therefore, a wide range of cancer cells may be safely targeted if the ligand-binding domain of this receptor is used in a construction of a chimeric antigen receptor (CAR) as an antigen recognition site, instead of using single chain variable domains derived from monoclonal antibody.We created several NKp44-based CAR constructs, which shares the extracellular NKp44 IG domain as a ligand-binding domain. Surface expression levels and subsequent functional properties can differ among T cells or NK cells transduced with novel CARs with different structural characteristics. We thus tested whether swapping the domains other than the antigen-binding domain affected expression and function. The CAR genes were retrovirally transduced into human primary T cells according to a standard method. We also transduced human primary NK cells with NKp44-based CARs, by a previously reported method (Imai C, et al. Blood 2005), to compare the expression pattern of the CAR in NK cells with that in human T cells.Retroviral transfer of wild type NKp44 gene and a construct harboring IC(p44) both did not induce NKp44 surface expression (Fig 1A,B). By sharp contrast, primary NK cells were able to express the CAR protein on the cell surface after transfer of these two genes. Removal of the IC(p44) [EH(p44)-TM(p44)-IC(CD3z)] allowed slight surface expression in T cells (Fig1C). The replacement of TM(p44) with TM(CD8a) resulted in higher surface expression in T cells (Fig 1D). These observations indicated the presence of IC(p44) as well as TM(p44) in the CAR constructs hampered surface expression in T cells most likely due to the lack of DAP12 expression. In addition to TM replacement, replacement of EH(p44) with EH(CD8a) markedly increased surface expression of the CAR (Fig 1E). Similarly, we tested use of CD28 domains instead of CD8a. Surprisingly, as different from the case of CD8a, the construct EH(p44)-TM(CD28)-IC(CD3z) yielded highest surface expression among the all CAR constructs created in this study in T cells as well as in NK cells (Fig1F), while the replacement of EH(p44) of the abovementioned CAR with EH(CD28) resulted in marked reduction of the CAR expression (Fig 1G).We confirmed surface expression of NKp44 ligand with flow cytometric analysis using recombinant human NKp44 Fc chimera protein (R&D Systems, McKinley Place, Minneapolis, USA) on various tumor cell lines including myeloid leukemia (K562, THP-1, U937, KY821, HL60), T-cell leukemia (PEER, MOLT4, HSB2), Burkitt lymphoma (Raji), BCR-ABL-positive B-ALL (OP-1), osteosarcoma (MG63, NOS1, NOS2, NOS10, U2OS, SaOS2), rhabdomyosarcoma (Rh28, RMS-YM), neuroblastoma (SK-N-SH, NB1, NB16, IMR32), and cervical carcinoma (Hela). Function of the best construct [EH(p44)-TM(CD28)-IC(CD3z)] was further evaluated. Primary T cells transduced with this NKp44-based CAR exerted powerful cytotoxicity against tumor cell lines tested and produced interferon-g and granzyme B, while GFP-transduced T cells and control T cells transduced with truncated NKp44-based CAR did not.In conclusion, we have created a novel CAR based on the antigen-binding property derived from NKp44 receptor immunoglobulin domain. This CAR should be effective to redirect T cells as well as NK cells against various types of cancer including hematological malignancies. [Display omitted] DisclosuresImai:Juno Therapeutics: Patents & Royalties.